David Bui Richard Lauhead Randall Mello Michelle Tran

•

Why is it important to have this kit?

Disregulation of the SUMO pathway has been linked to diseases including ovarian carcinoma, melanoma, and lung adenocarcinoma. (Mo and Moschos 2005)



Goal: Development of FRET based kit to screen compounds that could alter binding between SUMO1 and UBC9.

http://www.biochem.mpg.de/jentsch/Mueller.html

  Bradford Assay: Total protein concentrations of solution can be calculated using the equation obtained from graph. y = 2E-10x 1,2 1 0,8 0,6 3

Absorbance vs Protein amount

- 1E-06x 2 + 0.0018x - 0.0071

R² = 0.9991

abs Полиномиальная ( abs) 0,4 0,2 0 -0,2 0 500 1000 1500

Protein amount(ng)

2000 2500

 

Using highly pure proteins, serial dilutions were done to make solutions at different protein concentrations Values range from 1 ng to 100 ug

Ypet-Ubc9 Em 530 01/28/10 blank subtracted

70000000 60000000 50000000 y = 626,01x R² = 0,9847 40000000 Fluorescence(RFU) 30000000 20000000 10000000 0 Линейная ( Fluorescence(RFU)) -10000000 0 50000 100000

Protein Amount (ng)

150000

Ypet-Ubc9 Em 530 01/28/10 blank subtracted

70000000 60000000 50000000 y = 626,01x R² = 0,9847 40000000 30000000 20000000 10000000 0 -10000000 0 50000 100000

Protein Amount (ng)

150000 Fluorescence(RFU) Линейная ( Fluorescence(RFU))

  For accurate fluorescence measurements of single proteins and conjugations, an assay must have values away from background noise 500 ng is the lowest amount for usable assay conditions.

Ypet-Ubc9 (ng) 10000 RFU without blank RFU without blank RFU without blank 100000 58734866.98 55943707.39 61961881.18

50000 36425642.98 36053579.39 39276933.18

25000 20232692.98 20081895.39 23343669.18

7194076.48 7613824.887 9610900.176

5000 2500 1905796.605 1675790.887 1826577.176

1000 4313948.98 3672442.637 4147272.676

432355.386

416138.231

433805.832

500 118134.269

199872.325

247566.004

100 50 25 10 5 2.5

1 0 8604.365

2701.488

371.426

-1422.633

-1634.192

-1682.931

-1428.963

0 15551.569

17229.912

3043.334

-372.635

1552.071

2221.778

-136.699

0 13429.223

11205.072

8485.249

3125.487

1672.986

2313.75

965.361

0 Cypet-Sumo1 (ng) 2500 RFU without blank 493361.313

RFU without blank RFU without blank 91500 22667111.59 23441949.39 23460794.73

50000 9461644.594 13937961.39 15112699.73

25000 5334212.594 8311063.387 9603005.729

10000 2223233.344 2547688.137 4388946.729

5000 1010283.219 1294915.387 2053190.604

681590.324

730195.979

1000 500 135762.875

58105.11

196490.34

72562.403

217703.057

69732.471

100 50 6996.274

1396.175

6824.616

7010.968

7213.549

3055.906

25 10 5 2.5

1 0 5140.756

-1246.045

-1626.112

-1058.78

-465.929

0 3566.683

2060.112

3063.877

2058.266

13409.479

0 773.441

739.592

168.947

429.742

-50.309

0

• • Cell Lysate obtained from 1 Liter of solution and resuspended in 30 mL of Resuspension buffer was obtained.

Column purification protocol involves 10mL of lysate poured into a column with 500 uL of agarose nickel bead solution with subsequent 10 mL washes. • Elution took place 500 uL at a time and continued until the beads showed no fluorescence.

Ingredients Wash1 NaCL Tris HCL pH 7.4

Wash2 NaCL Tris HCL pH 7.4

Triton Wash3 NaCL Tris HCL pH 7.4

Immidazole Elution NaCL Tris HCL pH 7.4

Immidazole Resuspension Buffer Concentration(M) NaCl Tris-HCl pH 7.4

0.5

0.2

Immidazole

0.005

Protocol 1

0.3

0.02

Concentration of Solutions(M) Protocol 2

0.5

0.02

Protocol 3

0.4

0.02

0.3

0.2

0.50% 0.3

0.2

0.02

0.3

0.02

0.15

2 0.02

2.00% 2 0.02

0.05

0.3

0.02

0.25

1.2

0.02

1.25% 1.2

0.02

0.035

0.3

0.02

0.2

 Using the Bradford Assay to determine total protein concentration and the fluorescence curves generated from the sensitivity tests, the purity was calculated for each protocol Ypet-UBC9 Purification protocol Protocol1 Protocol2 Protocol3 Bradford concentrations(ng/uL) fluorescent concentration (ng/uL) Purity 7710 955 5500 5086.89

617.27

2783.92

0.66

0.65

0.51

Cypet-SUMO1 Purification protocol Protocol1 Protocol2 Protocol3 Bradford concentrations(ng/uL) 4844.94

fluorescent concentration (ng/uL) Purity 4708.36

0.97

3191.38

1678.81

0.53

4642.16

3229.12

0.70

Purity



Sensitivit y

(

Fluorescen t

Pr

otein

)

Bradford

(

Total

Pr

otein

)

• Ypet-UBC9 could be around 70% pure • Cypet-SUMO1 is unlikely to be 97% pure Ypet-UBC9 Cypet-SUMO1

• Keeping a constant fluorescent protein amount at 1 ug.

• BL21 cell lysate proteins were added to change percent purity.

• Results show that purity has little effect on fluorescence at 1ug of fluorescent protein.

Purity effects on Cypet-SUMO1 1ug

250000 200000 150000 100000 50000 0 450 470 490 510 530

Wavelength(nm)

550 570

Purity effects on Ypet-UBC9 1ug

700000 600000 500000 400000 300000 200000 100000 0 490 510 530 550 570

Wavelength(nm)

590 610 100% 90% 80% 70% 60% 50% 40% 30% 20% 100% 90% 80% 70% 60% 50% 40% 30% 20%

• • Tested purity effects on FRET with each protein at a constant amount of 500 ng. Results demonstrate that purity of fluorescent proteins and in FRET has no effect at low concentration(10ng/ul).

• Emission max of Ypet-Ubc9 over Cypet-SUMO1 to obtain FRET ratio.

• Results demonstrate little to no change in FRET ratio when purity is varied.

Purity effects on Fret 500ng

140000 120000 100000 80000 60000 40000 20000 0 450 470 490 510 530 550

Wavlength(nm)

570 590

Purity effect on Fret 500ng

610 1 0,8 0,6 0,4 0,2 0 0% 20% 40% 60%

Purity

80% 100% 120% 100% 90% 80% 70% 60% 50% 40% 30% 20% Ряд1

SUMO ASSAY KIT Tasks Purification optimization Flexstation 2 fluorescence sensitivity test Dialysis/Lyophilization Protein purity effects Expression optimization Start End 1/18/2010 2/8/2010 1/18/2010 2/8/2010 2/1/2010 2/22/2010 2/1/2010 2/22/2010 3/1/2010 3/15/2010 Fret sensitivity 3/1/2010 3/15/2010 compound screening sensitivity 3/22/2010 4/5/2010 Stability testing Oxidation testing Kit assembly Add secretion factors to proteins 3/22/2010 4/5/2010 4/12/2010 4/26/2010 4/12/2010 4/26/2010 5/3/2010 5/31/2010 %comple te 90% 100% 50%